Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.


Introduction
Pyruvate-ketal modified (henceforth termed "pyruvylated") glycans are found in various kingdoms of life where they have a wide repertoire of biological roles [1]. Pyruvylated galactose, for instance, is an epitope of the N-glycans of Schizosaccharomyces pombe [2], in the exopolysaccharide (EPS) of Xanthomonas campestris [3], and the capsular polysaccharides (CPS) of Bacteroides fragilis [4] and Streptococcus pneumoniae [5]. Pyruvylated N-acetylmannosamine (pyr-ManNAc) is present in peptidoglycan-linked cell wall glycopolymers (CWGPs) of Gram-positive bacteria, where it is an indispensable cell wall ligand for S-layer homology (SLH) domain-containing proteins [6,7]. Prominent examples ManNAc-GlcNAc-PP-UndPh. To learn about the substrate specificity of the CsaB enzyme, synthesised para-nitrophenyl-ManNAc and a methyl-glycoside were also tested as acceptors. The CsaB enzyme was produced as recombinant hexahistidine-tagged protein in E. coli and the formation of the pyruvylated product was confirmed by NMR spectroscopy. The kinetic constants of P. alvei CsaB under optimal reaction conditions-with regard to pH, temperature, time and concentration of Mg 2+ -including V max as well as K M and k cat values against the donor and the acceptor substrate were determined.
The recombinant enzymes were analysed by 10% SDS-PAGE in a Miniprotean™ apparatus (Biorad Hercules, CA, USA) according to Laemmli [23] upon Coomassie Brilliant Blue G250 (CBB) staining. The protein concentration was determined spectrophotometrically using the protein-specific extinction coefficient and molecular weight obtained from the exPASy ProtParam tool (http://web.expasy.org/protparam) (accessed from 1 March 2020 through 30 June 2021).

Enzymatic Preparation and Purification of β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh
Following the 1-h incubation at 37 • C, the reactions were incubated overnight at 25 • C to reach completeness. After stopping the reactions with 500 µL of ice-cold dH 2 O, the mixtures were loaded on a (C18) Sep-Pak classic cartridge (Waters; 36 mg sorbens) for selective binding of the lipid-like portion on the acceptor substrate. The rTagA reaction product (2) was eluted with 3 mL of MeOH (collecting 1.5-mL fractions in Eppendorf tubes), after prior removal of unbound material with 5 mL of dH 2 O. The purity of (2) after Sep-Pak purification was checked by 1 H NMR spectroscopy. For resalting of (2) from the Tris to the sodium form, 150 mg of Dowex 50XW8 (Na + ) cation exchange resin (VWR) was added per Eppendorf tube, and the tubes were shaken for 2 min and centrifuged (5000 rpm, 2 min). The supernatant, which was of neutral pH, was transferred into a Falcon tube (15 mL). The residual resin was washed three more times with ddH 2 O (2 mL, each), and all supernatants were combined, lyophilised and checked by 1 H NMR.

CsaB Activity Assay: Mode of Measurement
The Malachite Green Phosphate Assay Kit (Sigma-Aldrich, Cat No MAK307-1KT) is based on the colorimetric quantification of the green complex formed between Malachite Green, molybdate and free orthophosphate by measuring the absorbance value between 600 nm and 660 nm, according to the manufacturer's instructions.
To optimise the assay for the P. alvei pyruvyltransferase CsaB, which requires a noncommercial, difficult-to-access acceptor substrate (i.e., compound (2) from above) in conjunction with PEP as a donor, first, key assay parameters were evaluated using 5 µM NaH 2 PO 4 in dH 2 O as a test substance. Following the Assay Kit protocol, reagent A (Malachite Green oxalate and polyvinyl alcohol) and reagent B (ammonium molybdate in 3 M sulfuric acid) were mixed at a ratio of 100:1 (v/v) immediately before use and brought to 25 • C; 25 µL of this mixture (Malachite Green working reagent) was added to 100 µL of the 5 µM NaH 2 PO 4 solution in a 3-mm quartz cuvette and incubated for 30 min at 25 • C, as described in the manual, before measuring absorbance values over a range of 200-900 nm using a Hitachi U-3000 spectrophotometer to determine the optimal wavelength for quantifying the developed colour.
To determine the optimal temperature for colour development, the 5 µM NaH 2 PO 4 solution was incubated with reagent A and B at 4 • C, 25 • C and 37 • C for 30 min, each, followed by monitoring the absorbance values at the optimal wavelength as determined above over a period of 60 min.
To determine the potential influence of MgCl 2 as a possible CsaB activator on colour development, MgCl 2 was added to a final concentration of 0, 2, 5, 10, 30 or 100 mM.
A phosphate standard curve was generated by using a 0.1 M NaH 2 PO 4 solution with final concentrations of 0, 1, 2, 3, 6, 10, 20, 30 or 60 µM of NaH 2 PO 4 ; triplicate measurements were performed at 620 nm. The measured absorbance values of the standard substance were plotted and a linear regression was calculated.

CsaB Activity Assay: Reaction Parameters
For assaying the activity of CsaB on its known acceptor substrate (2) [11], key reaction parameters were analysed. All measurements were performed in triplicate and obtained absorbance values were blank-corrected by a control reaction without enzyme.
Based on a previously performed one-pot reaction [25], the PEP concentration was selected as 50 µM and the acceptor (2) concentration was 150 µM, which is below the K M , but due to its difficult synthesis it was not available in larger amounts. (2) was reacted with 50 µM PEP and 0.35 µg of purified rCsaB in a 100-µL reaction volume containing 10 mM MgCl 2 as a possible CsaB activator under different conditions of time, temperature and pH, followed by colour measurement in the 3-mm cuvette format.
The enzyme reaction was performed for 2, 5, 10 and 20 min and selected temperatures were 4 • C, 25 • C, 37 • C and 60 • C. The optimal pH was determined by performing the reaction in different buffers at a final concentration of 80 mM, including sodium citrate buffer (pH 4.0, pH 5.0), Tris-HCl (pH 6.0, pH 7.5) and Bis-Tris propane (pH 8.0, pH 9.0).
For structural confirmation of the rCsaB reaction product by NMR, the production of the pyruvylated product under optimal reaction conditions was upscaled using 1.5 mg (2).

Kinetic Analysis of CsaB with β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh Acceptor (2)
To determine the activity of rCsaB upon variation of PEP, eight data points were generated in triplicate using reactions containing 0.35 µg CsaB and 150 µM acceptor substrate (2) in a final volume of 100 µL of 80 mM Tris-HCl, pH 7.5. The PEP concentration was varied to reach a final concentration of 2, 4, 6, 10, 30, 50, 100 and 200 µM. To evaluate the possible influence of MgCl 2 on the enzyme velocity, the same reactions were performed with the addition of 10 mM MgCl 2 . After 5 min of incubation at 37 • C, the Malachite Green working reagent was added to stop the enzymatic reaction and the colour reaction was developed for 1 h at 37 • C followed by measuring the absorbance at 620 nm. Control reactions without enzyme were performed and data points were blank-corrected. Using the linear equation of the standard curve, the velocity was calculated by including the dilution factor of the enzyme and its concentration. Referring to the amount of rCsaB used in the assay, units (U) per minute were determined.
To estimate the activity of rCsaB upon variation of the acceptor substrate, seven data points were included, using reactions containing 0.35 µg rCsaB and 200 µM PEP (to ensure Biomolecules 2021, 11, 1732 6 of 15 pseudo-first-order conditions) in a final volume of 100 µL of 80 mM Tris-HCl, pH 7.5. The acceptor substrate concentration was varied, reaching a final concentration of 25, 50, 75, 100, 250, 500 and 1000 µM. The enzyme reaction, measurement and calculation of velocity were performed as described above.
The data were analysed using statistical software GraphPad Prism (version 9.1.2; GraphPad, San Diego, CA, USA), where K M and V max values were calculated by non-linear least-square regression to the direct Michaelis-Menten plot.

Testing of Alternate Substrates for CsaB
To analyse the suitability of alternate CsaB substrates, rCsaB was incubated with The standard mode of measurement as described above was used.

Expression and Purification of Recombinant Carbohydrate-Active Enzymes
To obtain ManNAc-containing acceptor substrates for the P. alvei CsaB enzyme, UDP-α-D-ManNAc is required. Since this compound is commercially unavailable, as an alternative to chemical synthesis, UDP-α-D-ManNAc was produced enzymatically from UDP-α-D-GlcNAc. For this purpose, the E. coli UDP-GlcNAc-2-epimerase WecB was produced recombinantly in E. coli BL21, enriched and purified using Superdex 200 SEC. In this way, rWecB (calculated molecular weight, 42.2 kDa) was obtained in high purity according to a CBB-stained 10% SDS-PAGE ( Figure S1).
The inverting UDP-ManNAc transferase TagA and the ketalpyruvyltransferase CsaB from the P. alvei CWGP biosynthesis pathway were freshly produced as recombinant proteins and purified by use of the translationally fused MBP and His 6 -tag, respectively [11].
Following either strategy, the reaction mixture was subjected to Sep-Pak purification [11], where (2) was recovered from the MeOH fraction as internal Tris salt in an already pure state. The Tris salt form was reflected by the presence of a characteristic multiplet at 3 ppm in the 1 H NMR spectrum ( Figure 1A). To avoid assay interference of the amino group of Tris, the Tris salt form of (2) was changed to the sodium salt via treatment with Dowex 50XW8 Na + form ion exchange resin, and the removal of Tris was confirmed via 1 H NMR, revealing the Tris signals to be absent ( Figure 1B). The overall yield of purified (2) in Na + form was 12 mg. tion towards (2) was monitored via H and P NMR spectroscopy [11].
Following either strategy, the reaction mixture was subjected to Sep-Pak purification [11], where (2) was recovered from the MeOH fraction as internal Tris salt in an already pure state. The Tris salt form was reflected by the presence of a characteristic multiplet at 3 ppm in the 1 H NMR spectrum ( Figure 1A). To avoid assay interference of the amino group of Tris, the Tris salt form of (2) was changed to the sodium salt via treatment with Dowex 50XW8 Na + form ion exchange resin, and the removal of Tris was confirmed via 1 H NMR, revealing the Tris signals to be absent ( Figure 1B). The overall yield of purified (2) in Na + form was 12 mg.  Table S1.

Set-Up of a Colorimetric CsaB Activity Assay
To optimise the colorimetric quantification of phosphate as a read-out for CsaB activity, which releases orthophosphate from the PEP substrate upon pyruvyltransfer, the Malachite Green Phosphate Assay Kit was used. Phosphate detection is based on the formation of a colour complex with Malachite Green and molybdate that is visible by a colour change from yellow-green to blue-green.

Set-Up of a Colorimetric CsaB Activity Assay
To optimise the colorimetric quantification of phosphate as a read-out for CsaB activity, which releases orthophosphate from the PEP substrate upon pyruvyltransfer, the Malachite Green Phosphate Assay Kit was used. Phosphate detection is based on the formation of a colour complex with Malachite Green and molybdate that is visible by a colour change from yellow-green to blue-green.
A wavelength scan between 200 and 900 nm revealed the absorbance maximum of the phosphomolybdate complex to be at 620 nm ( Figure 2A) and colour development being most intense after an incubation at 37 • C for 1 h (Figure 2B,C). Notably, a strong temperature dependence of the colour reaction was revealed by comparing the absorbance values at 4 • C, 25 • C and 37 • C ( Figure 2B). To test if MgCl 2 as a possible activator of CsaB interferes with colour development in the assay, several MgCl 2 concentrations were tested. It was found that the presence of MgCl 2 at a concentration above 2 mM reduced colour development, as evident from measuring the phosphate solution without enzyme ( Figure 2D). For maximum sensitivity, colour development in the CsaB pyruvyltransferase assay was measured at 620 nm after 1 h of colour complex formation at 37 • C. perature dependence of the colour reaction was revealed by comparing the absorbance values at 4 °C, 25 °C and 37 °C ( Figure 2B). To test if MgCl2 as a possible activator of CsaB interferes with colour development in the assay, several MgCl2 concentrations were tested. It was found that the presence of MgCl2 at a concentration above 2 mM reduced colour development, as evident from measuring the phosphate solution without enzyme ( Figure 2D). For maximum sensitivity, colour development in the CsaB pyruvyltransferase assay was measured at 620 nm after 1 h of colour complex formation at 37 °C. In an enzyme reaction set-up, where the release of phosphate from PEP and concomitant colour complex formation result from pyruvyltransfer to the saccharide acceptor ( Figure 3A), 150 µM ManNAc-GlcNAc-PP-UndPh acceptor (2) ( Figure 3B) and 50 µM PEP were reacted with 0.35 µg of rCsaB in buffered solution, and colour development was measured in a cuvette at 620 nm as determined above ( Figure 3C). In an enzyme reaction set-up, where the release of phosphate from PEP and concomitant colour complex formation result from pyruvyltransfer to the saccharide acceptor ( Figure 3A), 150 µM ManNAc-GlcNAc-PP-UndPh acceptor (2) ( Figure 3B) and 50 µM PEP were reacted with 0.35 µg of rCsaB in buffered solution, and colour development was measured in a cuvette at 620 nm as determined above ( Figure 3C).
The optimal incubation time to catch the enzyme in the linear range (initial rate) was determined to be 5 min, with the optima of pH and temperature being between pH 6.0 and 7.5, and 37 • C ( Figure 4A,B).
NMR analysis confirmed the structure of the pyruvylated rCsaB reaction product using (2) under the conditions defined above ( Figure 5). rCsaB retained full activity over a period of four weeks when stored at a concentration of 0.4 mg mL −1 in Tris/HCl, pH 7.5 at 4 • C. Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 15 The optimal incubation time to catch the enzyme in the linear range (initial rate) was determined to be 5 min, with the optima of pH and temperature being between pH 6.0 and 7.5, and 37 °C ( Figure 4A,B).  The optimal incubation time to catch the enzyme in the linear range (initial rate) was determined to be 5 min, with the optima of pH and temperature being between pH 6.0 and 7.5, and 37 °C ( Figure 4A,B). NMR analysis confirmed the structure of the pyruvylated rCsaB reaction product using (2) under the conditions defined above ( Figure 5).  [ 1 H, 13 C] Hetero multiple bond correlation (HMBC) spectra of the pyruvate C-2 region of the CsaB reaction product. The proton dimension is given in F2 while the carbon dimension is found in F1. The correlation between the pyruvate C-2 and the proton signals of the ManNAc H-6a and the pyruvate methyl group is in full agreement with [11] and shows the position of the introduced pyruvate group in acceptor (2). rCsaB retained full activity over a period of four weeks when stored at a concentration of 0.4 mg mL −1 in Tris/HCl, pH 7.5 at 4 °C.

Determination of Kinetic Constants for CsaB
Kinetic analysis revealed for rCsaB a KM, PEP value of 19.50 ± 3.50 µM, a kcat, PEP of 0.21 ± 0.01 s −1 and kcat/KM = 10.76 mM s −1 ( Figure 6A). Next, we determined if MgCl2, as a suggested enzyme activator [27], affects CsaB activity. The addition of 10 mM MgCl2 to the pyruvyltransferase reaction did not change the KM, PEP value (20.20 ± 2.70 µM) and marginally (but not significantly) decreased the kcat, PEP to 0.19 ± 0.01 s -1 ( Figure 6B). We conclude that MgCl2 is not necessary for CsaB activity. The KM for GlcNAc-ManNAc-PP-UndPh was determined to be 258 ± 38 µM (KM, Acceptor) with a kcat, Acceptor of 0.15 ± 0.01 s −1 and kcat/KM = 0.58 mM s −1 ( Figure 6C).  The proton dimension is given in F2 while the carbon dimension is found in F1. The correlation between the pyruvate C-2 and the proton signals of the ManNAc H-6a and the pyruvate methyl group is in full agreement with [11] and shows the position of the introduced pyruvate group in acceptor (2).

Determination of Kinetic Constants for CsaB
Kinetic analysis revealed for rCsaB a K M, PEP value of 19.50 ± 3.50 µM, a k cat, PEP of 0.21 ± 0.01 s −1 and k cat /K M = 10.76 mM s −1 ( Figure 6A). Next, we determined if MgCl 2 , as a suggested enzyme activator [27], affects CsaB activity. The addition of 10 mM MgCl 2 to the pyruvyltransferase reaction did not change the K M, PEP value (20.20 ± 2.70 µM) and marginally (but not significantly) decreased the k cat, PEP to 0.19 ± 0.01 s −1 ( Figure 6B). We conclude that MgCl 2 is not necessary for CsaB activity. The K M for GlcNAc-ManNAc-PP-UndPh was determined to be 258 ± 38 µM (K M, Acceptor ) with a k cat, Acceptor of 0.15 ± 0.01 s −1 and k cat /K M = 0.58 mM s −1 ( Figure 6C). [ 1 H, 13 C] Hetero multiple bond correlation (HMBC) spectra of the pyruvate C-2 region of the CsaB reaction product. The proton dimension is given in F2 while the carbon dimension is found in F1. The correlation between the pyruvate C-2 and the proton signals of the ManNAc H-6a and the pyruvate methyl group is in full agreement with [11] and shows the position of the introduced pyruvate group in acceptor (2). rCsaB retained full activity over a period of four weeks when stored at a concentration of 0.4 mg mL −1 in Tris/HCl, pH 7.5 at 4 °C.

Testing Alternate Substrates for CsaB
Providing 1-O Me glycoside (4) or pNP-β-D-ManNAc (5) as potential acceptor substrates in a kinetics CsaB reaction set-up without MgCl 2 , the detectable colour development did not exceed that of the control reaction without enzyme (Figure 7). This implies that neither of these compounds is a suitable acceptor substrate for P. alvei CsaB.

Testing Alternate Substrates for CsaB
Providing 1-O Me glycoside (4) or pNP-β-D-ManNAc (5) as potential acceptor substrates in a kinetics CsaB reaction set-up without MgCl2, the detectable colour development did not exceed that of the control reaction without enzyme (Figure 7). This implies that neither of these compounds is a suitable acceptor substrate for P. alvei CsaB.

Discussion
Pyruvyltransferases are widespread in nature; they occur in almost all bacterial phyla, several yeast species and in algae, but not in humans [1]. They catalyse pyruvate formation as a biologically potent non-carbohydrate modification of various glycoconjugates [1] and are promising anti-infective targets. The enol-pyruvyltransferase MurA from the bacterial peptidoglycan biosynthesis pathway, for instance, imparts fosfomycin resistance and is currently under evaluation towards new inhibitors [19]. According to the World Health Organization, 750,000 deaths per year are caused by antibiotic-resistant bacteria, and the rise of antibiotic resistances necessitates alternate strategies to counteract bacterial infections. Traditionally, the bacterial cell wall is a prominent target point for antimicrobial agents. For instance, one Achilles heel of the methicillin-resistant superbug Staphylococcus aureus is its cell wall teichoic acid; if enzymes within its biosynthesis pathway are disrupted, β-lactam antibiotic sensitivity is restored and host colonisation is impaired [28,29].
A 4,6-ketalpyruvylated β-D-ManNAc residue as an integral part of various bacterial CWGPs is crucial for sticking the Gram-positive cell wall together [6,9,30]. B. anthracis is the most prominent example of a pathogen that has integrated the pyr-ManNAc epitope into its cell wall building plan [9]. To uncover details of ManNAc pyruvylation, the honeybee saprophyte P. alvei serves as an ideal model due to analogies of CWGP composition and overall cell wall architecture with B. anthracis. We have previously identified the CsaB enzyme encoded in the P. alvei CWGP biosynthesis gene cluster as a 4,6-ketalpyruvyl::ManNAc transferase that is active on the synthetic CWGP biosynthesis precursor analogue β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh (2) [11]. A CsaB homologue is also encoded in the B. anthracis CWGP biosynthesis gene locus [31].
Currently, there is no fast and straightforward assay for the measurement of the activity and kinetics of pyruvyltransferases available. To circumvent the requirement of labelled acceptor substrates for on-column detection and isolation of pyruvylated reaction products, the Malachite Green Phosphate Assay [18][19][20][21] was optimised for assaying ketalpyruvyltransfer to synthetic saccharide acceptor substrates, exemplified with P. alvei CsaB. The assay with a sensitivity range of 0.02 to 40 µM phosphate (according to the manufacturer) is based on phosphate release during the pyruvyltransfer reaction due to the splitting of PEP into a pyruvate entity and inorganic phosphate followed by a colour

Discussion
Pyruvyltransferases are widespread in nature; they occur in almost all bacterial phyla, several yeast species and in algae, but not in humans [1]. They catalyse pyruvate formation as a biologically potent non-carbohydrate modification of various glycoconjugates [1] and are promising anti-infective targets. The enol-pyruvyltransferase MurA from the bacterial peptidoglycan biosynthesis pathway, for instance, imparts fosfomycin resistance and is currently under evaluation towards new inhibitors [19]. According to the World Health Organization, 750,000 deaths per year are caused by antibiotic-resistant bacteria, and the rise of antibiotic resistances necessitates alternate strategies to counteract bacterial infections. Traditionally, the bacterial cell wall is a prominent target point for antimicrobial agents. For instance, one Achilles heel of the methicillin-resistant superbug Staphylococcus aureus is its cell wall teichoic acid; if enzymes within its biosynthesis pathway are disrupted, β-lactam antibiotic sensitivity is restored and host colonisation is impaired [28,29].
A 4,6-ketalpyruvylated β-D-ManNAc residue as an integral part of various bacterial CWGPs is crucial for sticking the Gram-positive cell wall together [6,9,30]. B. anthracis is the most prominent example of a pathogen that has integrated the pyr-ManNAc epitope into its cell wall building plan [9]. To uncover details of ManNAc pyruvylation, the honeybee saprophyte P. alvei serves as an ideal model due to analogies of CWGP composition and overall cell wall architecture with B. anthracis. We have previously identified the CsaB enzyme encoded in the P. alvei CWGP biosynthesis gene cluster as a 4,6-ketalpyruvyl::ManNAc transferase that is active on the synthetic CWGP biosynthesis precursor analogue β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh (2) [11]. A CsaB homologue is also encoded in the B. anthracis CWGP biosynthesis gene locus [31].
Currently, there is no fast and straightforward assay for the measurement of the activity and kinetics of pyruvyltransferases available. To circumvent the requirement of labelled acceptor substrates for on-column detection and isolation of pyruvylated reaction products, the Malachite Green Phosphate Assay [18][19][20][21] was optimised for assaying ketalpyruvyltransfer to synthetic saccharide acceptor substrates, exemplified with P. alvei CsaB. The assay with a sensitivity range of 0.02 to 40 µM phosphate (according to the manufacturer) is based on phosphate release during the pyruvyltransfer reaction due to the splitting of PEP into a pyruvate entity and inorganic phosphate followed by a colour reaction. Notably, absorbance values of the formed colour complex measured in microtiter plates in the Tecan plate reader were generally lower and noisier than those measured spectrophotometrically in a 3-mm quartz cuvette (F.F. Hager-Mair, C. Stefanović, data not shown). For this reason, the enzyme assay was optimised for the cuvette format. In the colour reaction, we obtained high background values in control reactions without enzyme, using β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh (2) as a CsaB acceptor substrate, which we initially attributed to the complexity of the lipid-like tail of (2); lipids were described in the Sigma-Aldrich manual to interfere with the Malachite Green dye, with the chemistry behind it unknown. Surprisingly, the use of methanol, ethanol or propanol in a control reaction yielded a comparably high background signal. Thus, it is imperative to perform the full range of control reactions when assaying pyruvyltransferases with the Malachite Green Phosphate Assay in order to avoid false positive results. Furthermore, given the strong temperature dependence of the colour reaction ( Figure 2B), the temperature should be tightly controlled to obtain reliable results. Notably, these findings might also be of relevance when using the Malachite Green Assay Kit for investigating other enzymes that release inorganic phosphate.
As defined within this study, an optimal assay set-up for quantifying phosphate release upon pyruvyltransfer catalysed by P. alvei CsaB contains 0.35 µg of recombinant enzyme, 200 µM PEP and an acceptor concentration above K M (250-1000 µM, if enough acceptor is available), with colour development for 60 min and measurement in a 3-mm quartz cuvette at 620 nm. Notably, a shortage of synthesised acceptor (2) precluded its use at a concentration ensuring a saturating concentration in the assays. rCsaB was determined to have optimal activity at 37 • C and pH 7.5. The addition of MgCl 2 to the assay had no significant effect on the catalytic activity of P. alvei CsaB ( Figure 6A,B). Notably, MgCl 2 above a concentration of 2 mM seems to decrease colour development in the Malachite Green Phosphate Assay under the chosen conditions, possibly due to interference with assay reagents or phosphate or PEP (compare with Figure 2D) [32]. Notably, for the yeast pyruvyltransferase Pvg1p, an inhibitory effect of Co 2+ , Ni 2+ and Cd 2+ was reported [15]; in that study, however, Mg 2+ was not included.
Using the reaction set-up defined within the frame of this study, kinetic constants could be determined for rCsaB by fitting the data to the Michaelis-Menten equation (Table 1). This revealed a K M value for the PEP donor substrate of 19.50 ± 3.5 µM, which is~10-fold lower compared to the K M, PEP reported for the B. fragilis CPS 4,6-ketalpyruvl::galactose transferase WcfO [4], suggesting a higher affinity of CsaB for the donor substrate. Of note, K M, PEP values of 199 µM, 121 µM and of 0.4 µM were reported for two mycobacterial UDP-N-acetylglucosamine enolpyruvyl transferases (M. tuberculosis MurA and M. smegmatis MurA) and for E. coli MurA, respectively [18,33]. Furthermore, our study presents the first kinetic constants of a pyruvyltransferase towards the acceptor substrate, with K M, Acceptor = 258.00 ± 38.00 µM and a k cat, Acceptor of 0.15 ± 0.01 s −1 as determined for CsaB towards β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh (2). The K M, Acceptor is 13 times higher than the K M, PEP , indicating that the affinity of CsaB for PEP is higher than for the acceptor.   [33] The failure of CsaB to catalyse ketalpyruvyltransfer to both pNP-β-D-ManNAc and β-D-ManNAc-(1→4)-β-D-GlcNAc-1-OMe (3) supports the necessity of a lipid-like tail and/or phosphate on a suitable CsaB substrate. This assumption is in agreement with data obtained with B. fragilis WcfO, which was found to be active on a lipid-bound tetrasaccharide CPS repeat [4], and with the predicted CWGP acceptor substrate for B. anthracis CsaB [31].
The developed enzyme assay is crucial for future mechanistic studies by the use of rationally designed pyruvyltransferases as well as for future inhibitor design to combat bacterial pathogens.